Method for treating diseases using HSP90-inhibiting agents in combination with antimetabolites

ABSTRACT

The present invention provides a method for treating cancer. The method involves the administration of an HSP90 inhibitor and an antimetabolite, where the combined administration provides a synergistic effect. In one aspect of the invention, a method of treating cancer is provided where a subject is treated with a dose of an HSP90 inhibitor in one step and a dose of an antimetabolite in another step. In another aspect of the invention, a method of treating cancer is provided where a subject is first treated with a dose of an HSP90 inhibitor and subsequently treated with a dose of an antimetabolite. In another aspect of the invention, a method of treating cancer is provided where a subject is first treated with a dose of an antimetabolite and subsequently treated with a dose of an HSP90 inhibitor.

CROSS REFERENCE TO RELATED U.S. PATENT APPLICATIONS

The present application claims the benefit of Provisional Patent Application No. 60/474,906, which was filed May 30, 2003, under 35 U.S.C. § 119(e). The provisional application is hereby incorporated-by-reference into this application for all purposes.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

This invention relates to methods for treating cancer in which an inhibitor of Heat Shock Protein 90 (“HSP90”) is combined with an antimetabolite. More particularly, this invention relates to combinations of the HSP90 inhibitor geldanamycin and its derivatives, especially 17-alkylamino-17-desmethoxygeldanamycin (“17-AAG”) and 17-(2-dimethylaminoethyl)amino-17-desmethoxygeldanamycin (“17-DMAG”), with an antimetabolite (e.g., 5-fluorouracil and gemcitabine).

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DISCUSSION

Geldanamycin (figure below, R₁₇═—OCH₃) is a benzoquinone ansamycin polyketide isolated from Streptomyces geldanus. Although originally discovered by screening microbial extracts for antibacterial and antiviral activity, geldanamycin was later found to be cytotoxic to certain tumor cells in vitro and to reverse the morphology of cells transformed by the Rous sarcoma virus to a normal state.

Geldanamycin's nanomolar potency and apparent specificity for aberrant protein kinase dependent tumor cells, as well as the discovery that its primary target in mammalian cells is the ubiquitous Hsp90 protein chaperone, has stimulated interest in the development of this compound as an anti-cancer drug. However, the association of unacceptable hepatotoxicity with the administration of geldanamycin led to its withdrawal from Phase I clinical trials.

More recently, attention has focused on 17-amino derivatives of geldanamycin, in particular 17-(allylamino)-17-desmethoxygeldanamycin (“17-AAG”, R₁₇═—NCH₂CH═CH₂). This compound has reduced hepatotoxicity while maintaining useful Hsp90 binding. Certain other 17-amino derivatives of geldanamycin, 11-oxogeldanamycin, and 5,6-dihydrogeldanamycin, are disclosed in U.S. Pat. Nos. 4,261,989, 5,387,584 and 5,932,566, each of which is incorporated herein by reference. Treatment of cancer cells with geldanamycin or 17-AAG causes a retinoblastoma protein-dependent G1 block, mediated by down-regulation of the induction pathways for cyclin D-cyclin dependent cdk4 and cdk6 protein kinase activity. Cell cycle arrest is followed by differentiation and apoptosis. G1 progression is unaffected by geldanamycin or 17-AAG in cells with mutated retinoblastoma protein; these cells undergo cell cycle arrest after mitosis, again followed by apoptosis.

The above-described mechanism of geldanamycin and 17-AAG appears to be a common mode of action among the benzoquinone ansamycins that further includes binding to Hsp90 and subsequent degradation of Hsp90-associated client proteins. Among the most sensitive client protein targets of the benzoquinone ansamycins are the Her kinases (also known as ErbB), Raf, Met tyrosine kinase, and the steroid receptors. Hsp90 is also involved in the cellular response to stress, including heat, radiation, and toxins. Certain benzoquinone ansamycins, such as 17-AAG, have thus been studied to determine their interaction with cytotoxins that do not target Hsp90 client proteins.

U.S. Pat. Nos. 6,245,759, 6,306,874 and 6,313,138, each of which is incorporated herein by reference, disclose compositions comprising certain tyrosine kinase inhibitors together with 17-AAG and methods for treating cancer with such compositions. Münster, et al., “Modulation of Hsp90 function by ansamycins sensitizes breast cancer cells to chemotherapy-induced apoptosis in an RB- and schedule-dependent manner,” Clinical Cancer Research (2001) 7:2228-2236, discloses that 17-AAG sensitizes cells in culture to the cytotoxic effects of Paclitaxel and doxorubicin. The Munster reference further discloses that the sensitization towards paclitaxel by 17-AAG is schedule-dependent in retinoblastoma protein-producing cells due to the action of these two drugs at different stages of the cell cycle: treatment of cells with a combination of paclitaxel and 17-AAG is reported to give synergistic apoptosis, while pretreatment of cells with 17-AAG followed by treatment with paclitaxel is reported to result in abrogation of apoptosis. Treatment of cells with paclitaxel followed by treatment with 17-AAG 4 hours later is reported to show a synergistic effect similar to coincident treatment.

Citri, et al., “Drug-induced ubiquitylation and degradation of ErbB receptor tyrosine kinases: implications for cancer chemotherapy,” EMBO Journal (2002) 21:2407-2417, discloses an additive effect upon co-administration of geldanamycin and an irreversible protein kinase inhibitor, CI-1033, on growth of ErbB2-expressing cancer cells in vitro. In contrast, an antagonistic effect of CI-1033 and anti-ErB2 antibody, Herceptin is disclosed.

Thus, while there has been a great deal of research interest in the benzoquinone ansamycins, particularly geldanamycin and 17-AAG, there remains a need for effective therapeutic regimens to treat cancer or other disease conditions characterized by undesired cellular hyperproliferation using such compounds, whether alone or in combination with other agents.

SUMMARY OF THE INVENTION

The present invention provides a method for treating cancer. The method involves the administration of an HSP90 inhibitor and an antimetabolite, where the combined administration provides a synergistic effect.

In one aspect of the invention, a method of treating cancer is provided where a subject is treated with a dose of an HSP90 inhibitor in one step and a dose of an antimetabolite in another step.

In another aspect of the invention, a method of treating cancer is provided where a subject is first treated with a dose of an HSP90 inhibitor and subsequently treated with a dose of an antimetabolite.

In another aspect of the invention, a method of treating cancer is provided where a subject is first treated with a dose of an antimetabolite and subsequently treated with a dose of an HSP90 inhibitor.

In another aspect of the invention, a method of treating cancer is provided where a subject is first treated with a dose of an antimetabolite (e.g., 5-fluorouracil or gemcitabine). After waiting for a period of time sufficient to allow development of a substantially efficacious response of the antimetabolite, a formulation comprising a synergistic dose of a benzoquinone ansamycin together with a second sub-toxic dose of the antimetabolite is administered.

In another aspect of the invention, a method of treating cancer is provided where a subject is treated first with a dose of a benzoquinone ansamycin, and second, a dose of an antimetabolite. After waiting for a period of time sufficient to allow development of a substantially efficacious response of the antimetabolite, a formulation comprising a synergistic dose of a benzoquinone ansamycin together with a second sub-toxic dose of the oncolytic drug is administered.

In another aspect of the invention, a method for treating cancer is provided where a subject is treated with a dose of an HSP90 inhibitor in one step and a dose of an antimetabolite in another step, and where a side effect profile for the combined, administered drugs is substantially better than for the antimetabolite alone.

In another aspect of the invention, a method for treating colorectal cancer is provided where a subject is treated with a dose of an HSP90 inhibitor in one step and a dose of an antimetabolite in another step. The HSP90 inhibitor for this aspect is typically 17-AAG, while the antimetabolite is usually either 5-fluorouracil or gemcitabine. Where the antimetabolite is 5-fluorouracil, it is typically administered before the 17-AAG; where the antimetabolite is gemcitabine, it is typically administered after the 17-AAG.

DEFINITIONS

“Antimetabolite” refers to an antineoplastic drug that inhibits the utilization of a metabolite or a prodrug thereof. Examples of antimetabolites include, without limitation, methotrexate, 5-fluorouracil, 5-fluorouracil prodrugs (e.g., capecitabine), 5-fluorodeoxyuridine monophosphate, cytarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine.

“HSP90 inhibitor” refers to a compound that inhibits the activity of heat shock protein 90, which is a cellular protein responsible for chaperoning multiple client proteins necessary for cell signaling, proliferation and survival. One class of HSP90 inhibitors is the benzoquinone ansamycins. Examples of such compounds include, without limitation, geldanamycin and geldanamycin derivatives (e.g., 17-alkylamino-17-desmethoxy-geldanamycin (“17-AAG”) and 17-(2-dimethylaminoethyl)amino-17-desmethoxy-geldanamycin (“17-DMAG”)). See Sasaki et al., U.S. Pat. No. 4,261,989 (1981) for synthesis of 17-AAG and Snader et al., U.S. 2004/0053909 A1 (2004) for synthesis of 17-DMAG). In addition to 17-AAG and 17-DMAG, other preferred geldanamycin derivatives are 11-O-methyl-17-(2-(1-azetidinyl)ethyl)amino-17-demethoxygeldanamycin (A), 11-O-methyl-17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (B), and 11-O-methyl-17-(2-(1-pyrrolidinyl)ethyl)amino-17-demethoxygeldanamycin (C), whose synthesis is described in the co-pending commonly U.S. patent application of Tian et al., Ser. No. 10/825,788, filed Apr. 16, 2004, and in Tian et al., PCT application no. PCT/US04/11638, filed Apr. 16, 2004; the disclosures of which are incorporated herein by reference. Additional preferred geldanamycin derivatives are described in Santi et al., U.S. 2003/0114450 A1 (2003), also incorporated by reference.

“MTD” refers to maximum tolerated dose. The MTD for a compound is determined using methods and materials known in the medical and pharmacological arts, for example through dose-escalation experiments. One or more patients is first treated with a low dose of the compound, typically about 10% of the dose anticipated to be therapeutic based on results of in vitro cell culture experiments. The patients are observed for a period of time to determine the occurrence of toxicity. Toxicity is typically evidenced as the observation of one or more of the following symptoms: vomiting, diarrhea, peripheral neuropathy, ataxia, neutropenia, or elevation of liver enzymes. If no toxicity is observed, the dose is increased about 2-fold, and the patients are again observed for evidence of toxicity. This cycle is repeated until a dose producing evidence of toxicity is reached. The dose immediately preceding the onset of unacceptable toxicity is taken as the MTD.

“Side effects” refer to a number of toxicities typically seen upon treatment of a subject with an antineoplastic drug. Such toxicities include, without limitation, anemia, anorexia, bilirubin effects, dehydration, dermatology effects, diarrhea, dizziness, dyspnea, edema, fatigue, headache, hematemesis, hypokalemia, hypoxia, musculoskeletal effects, myalgia, nausea, neuro-sensory effects, pain, rash, serum glutamic oxaloacetic transaminase effects, serum glutamic pyruvic transaminase effects, stomatitis, sweating, taste effects, thrombocytopenia, voice change, and vomiting.

“Side effect grading” refers to National Cancer Institute common toxicity criteria (NCI CTC, Version 2). Grading runs from 1 to 4, with a grade of 4 representing the most serious toxicities.

COMBINATION THERAPY

The present invention provides a method for treating cancer. The method involves the administration of an HSP90 inhibitor and an antimetabolite, where the combined administration provides a synergistic effect.

Suitable HSP90 inhibitors used in the present invention include benzoquinone ansamycins. Typically, the benzoquinone ansamycin is geldanamycin or a geldanamycin derivative. Preferably, the benzoquinone ansamycin is a geldanamycin derivative selected from a group consisting of 17-alkylamino-17-desmethoxy-geldanamycin (“17-AAG”), 17-(2-dimethylaminoethyl)amino-17-desmethoxy-geldanamycin (“17-DMAG”), 11-O-methyl-17-(2-(1-azetidinyl)ethyl)amino-17-demethoxygeldanamycin, 11-O-methyl-17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin, and 11-O-methyl-17-(2-(1-pyrrolidinyl)ethyl)amino-17-demethoxygeldanamycin.

Antimetabolites employed in the present method include, without limitation, methotrexate, 5-fluorouracil, 5-fluorouracil prodrugs (e.g., capecitabine), 5-fluorodeoxyuridine monophosphate, cytarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Preferably, the antimetabolite is 5-fluorouracil or gemcitabine.

The dose of antimetabolite used as a partner in combination therapy with an HSP90 inhibitor (e.g., benzoquinone ansamycin) is determined based on the maximum tolerated dose observed when the antimetabolite is used as the sole therapeutic agent. In one embodiment of the invention, the dose of antimetabolite when used in combination therapy with a benzoquinone ansamycin is the MTD. In other embodiments of the invention, the dose of antimetabolite when used in combination therapy with a benzoquinone ansamycin is between about 1% of the MTD and the MTD, between about 5% of the MTD and the MTD, between about 5% of the MTD and 75% of the MTD, or between about 25% of the MTD and 75% of the MTD.

Use of the benzoquinone ansamycin allows for use of a lower therapeutic dose of an antimetabolite, thus significantly widening the therapeutic window for treatment. In one embodiment, the therapeutic dose of antimetabolite is lowered by at least about 10%. In other embodiments the therapeutic dose is lowered from about 10 % to 20%, from about 20% to 50%, from about 50% to 200%, or from about 100% to 1,000%.

For the treatment of a variety of carcinomas, the recommended dose of the antimetabolite 5-fluorouracil for an average patient is 12 mg/kg daily for 4 days, by rapid injection, followed by 6 mg/kg on alternate succeeding days for four doses if no toxicity is observed. An arbitrary maximal daily dose for the preceding regimen has been set at 800 mg. Other regimens use daily doses of 500 mg/m² 5-fluorouracil for 5 days, repeated in monthly cycles. For the antimetabolite gemcitabine, the administered dose is typically 1,000 to 1,500 mg/m² over 30 min once a week.

The synergistic dose of the benzoquinone ansamycin used in combination therapy is determined based on the maximum tolerated dose observed when the benzoquinone ansamycin is used as the sole therapeutic agent. Clinical trials have determined an MTD for 17-AAG of about 40 mg/m² utilizing a daily ×5 schedule, an MTD of about 220 mg/m² utilizing a twice-weekly regimen, and an MTD of about 308 mg/m² utilizing a once-weekly regimen. In one embodiment of the invention, the dose of the benzoquinone ansamycin when used in combination therapy is the MTD. In another embodiment of the invention, the does of the benzoquinone ansamycin when used in combination therapy is between about 1% of the MTD and the MTD, between about 5% of the MTD and the MTD, between about 5% of the MTD and 75% of the MTD, or between about 25% of the MTD and 75% of the MTD.

Where the benzoquinone ansamycin is 17-AAG, and the administration of compound is weekly, its therapeutic dose is typically between 50 mg/m² and 450 mg/m². Preferably, the dose is between 150 mg/m² and 350 mg/m², and about 308 mg/m² is especially preferred. Where the administration of compound is biweekly (i.e., twice per week), the therapeutic dose of 17-AAG is typically between 50 mg/m² and 250 mg/m². Preferably, the dose is between 150 mg/m² and 250 mg/m², and about 220 mg/m² is especially preferred.

Where the present method involves the administration of 17-AAG and 5-fluorouracil, a dosage regimen involving one or more administrations of the combination per week is typical. Oftentimes, the dosage regimen involves 2, 3, 4 or 5 administrations per week. Tables 1 and 2 below show a number of 5-fluorouracil/17-AAG dosage combinations (i.e., dosage combinations 0001 to 0096). TABLE 1 5-Fluorouracil/17-AAG dosage combinations. 30-100 100-150 150-200 mg/m² mg/m² mg/m² 200-250 mg/m² 17-AAG 17-AAG 17-AAG 17-AAG  0-1 mg/m² 0001 0002 0003 0004 5-fluorouracil  1-2 mg/m² 0005 0006 0007 0008 5-fluorouracil  2-3 mg/m² 0009 0010 0011 0012 5-fluorouracil  3-4 mg/m² 0013 0014 0015 0016 5-fluorouracil  4-5 mg/m² 0017 0018 0019 0020 5-fluorouracil  5-6 mg/m² 0021 0022 0023 0024 5-fluorouracil  6-7 mg/m² 0025 0026 0027 0028 5-fluorouracil  7-8 mg/m² 5- 0029 0030 0031 0032 fluorouracil  8-9 mg/m² 0033 0034 0035 0036 5-fluorouracil  9-10 mg/m² 0037 0038 0039 0040 5-fluorouracil 10-11 mg/m² 0041 0042 0043 0044 5-fluorouracil 11-12 mg/m² 0045 0046 0047 0048 5-fluorouracil

TABLE 2 5-Fluorouracil/17-AAG dosage combinations continued. 250-300 300-350 mg/m² mg/m² 350-400 mg/kg 400-450 mg/kg 17-AAG 17-AAG 17-AAG 17-AAG  0-1 mg/m² 0049 0050 0051 0052 5-fluorouracil  1-2 mg/m² 0053 0054 0055 0056 5-fluorouracil  2-3 mg/m² 0057 0058 0059 0060 5-fluorouracil  3-4 mg/m² 0061 0062 0063 0064 5-fluorouracil  4-5 mg/m² 0065 0066 0067 0068 5-fluorouracil  5-6 mg/m² 0069 0070 0071 0072 5-fluorouracil  6-7 mg/m² 0073 0074 0075 0076 5-fluorouracil  7-8 mg/m² 0077 0078 0079 0080 5-fluorouracil  8-9 mg/m² 0081 0082 0083 0084 5-fluorouracil  9-10 mg/m² 0085 0086 0087 0088 5-fluorouracil 10-11 mg/m² 0089 0090 0091 0092 5-fluorouracil 11-12 mg/m² 0093 0094 0095 0096 5-fluorouracil

Where the present method involves the administration of 17-AAG and gemcitabine, a dosage regimen involving one or two administrations of the combination per week is typical. Tables 3 and 4 below show a number of gemcitabine/17-AAG dosage combinations (i.e., dosage combinations 0097 to 0212). TABLE 3 Gemcitabine/17-AAG dosage combinations. 100-150 150-200 200-250 30-100 mg/m² mg/m² mg/m² mg/m² 17-AAG 17-AAG 17-AAG 17-AAG   0-100 mg/m² 0097 0098 0099 0100 gemcitabine  100-200 mg/m² 0101 0102 0103 0104 gemcitabine  200-300 mg/m² 0105 0106 0107 0108 gemcitabine  300-400 mg/m² 0109 0110 0111 0112 gemcitabine  400-500 mg/m² 0113 0114 0115 0116 gemcitabine  500-600 mg/m² 0117 0118 0119 0120 gemcitabine  600-700 mg/m² 0121 0122 0123 0124 gemcitabine  700-800 mg/m² 0125 0126 0127 0128 gemcitabine  800-900 mg/m² 0129 0130 0131 0132 gemcitabine  900-1000 mg/m² 0133 0134 0135 0136 gemcitabine 1000-1100 mg/m² 0137 0138 0139 0140 gemcitabine 1100-1200 mg/m² 0141 0142 0143 0144 gemcitabine 1200-1300 mg/m² 0145 0146 0147 0148 gemcitabine 1300-1400 mg/m² 0149 0150 0151 0152 gemcitabine 1400-1500 mg/m² 0153 0154 0155 0156 gemcitabine

TABLE 4 Gemcitabine/17-AAG dosage combinations continued. 250-300 300-350 350-400 400-450 mg/m² mg/m² mg/kg mg/kg 17-AAG 17-AAG 17-AAG 17-AAG   0-100 mg/m² 0153 0154 0155 0156 gemcitabine  100-200 mg/m² 0157 0158 0159 0160 gemcitabine  200-300 mg/m² 0161 0162 0163 0164 gemcitabine  300-400 mg/m² 0165 0166 0167 0168 gemcitabine  400-500 mg/m² 0169 0170 0171 0172 gemcitabine  500-600 mg/m² 0173 0174 0175 0176 gemcitabine  600-700 mg/m² 0177 0178 0179 0180 gemcitabine  700-800 mg/m² 0181 0182 0183 0184 gemcitabine  800-900 mg/m² 0185 0186 0187 0188 gemcitabine  900-1000 mg/m² 0189 0190 0191 0192 gemcitabine 1000-1100 mg/m² 0193 0194 0195 0196 gemcitabine 1100-1200 mg/m² 0197 0198 0199 0200 gemcitabine 1200-1300 mg/m² 0201 0202 0203 0204 gemcitabine 1300-1400 mg/m² 0205 0206 0207 0208 gemcitabine 1400-1500 mg/m² 0209 0210 0211 0212 gemcitabine

The method of the present invention may be carried out in at least two basic ways. A subject may first be treated with a dose on an HSP90 inhibitor and subsequently be treated with a dose of an antimetabolite. Alternatively, the subject may first be treated with a dose of an antimetabolite and subsequently be treated with a dose of an HSP90 inhibitor. The appropriate dosing regimen depends on the particular antimetabolite employed.

In another aspect of the invention, a subject is first treated with a dose of an antimetabolite (e.g., 5-fluorouracil or gemcitabine). After waiting for a period of time sufficient to allow development of a substantially efficacious response of the oncolytic drug, a formulation comprising a synergistic dose of a benzoquinone ansamycin together with a second sub-toxic dose of the antimetabolite is administered. In general, the appropriate period of time sufficient to allow development of a substantially efficacious response to the antimetabolite will depend upon the pharmacokinetics of the antimetabolite, and will have been determined during clinical trials of therapy using the antimetabolite alone. In one embodiment of the invention, the period of time sufficient to allow development of a substantially efficacious response to the antimetabolite is between about 1 hour and 96 hours. In another aspect of the invention, the period of time sufficient to allow development of a substantially efficacious response to the antimetabolite is between about 2 hours and 48 hours. In another embodiment of the invention, the period of time sufficient to allow development of a substantially efficacious response to the antimetabolite is between about 4 hours and 24 hours.

In another aspect of the invention, a subject is treated first with one of the above-described benzoquinone ansamycins, and second, a dose of an antimetabolite, such as, but not limited to, 5-fluorouracil and gemcitabine. After waiting for a period of time sufficient to allow development of a substantially efficacious response of the antimetabolite, a formulation comprising a synergistic dose of a benzoquinone ansamycin together with a second sub-toxic dose of the antimetabolite is administered. In general, the appropriate period of time sufficient to allow development of a substantially efficacious response to the antimetabolite will depend upon the pharmacokinetics of the antimetabolite, and will have been determined during clinical trials of therapy using the antimetabolite alone. In one embodiment of the invention, the period of time sufficient to allow development of a substantially efficacious reponse to the antimetabolite is between about 1 hour and 96 hours. In another aspect of the invention, the period of time sufficient to allow development of a substantially efficacious response to the antimetabolite is between about 2 hours and 48 hours. In another embodiment of the invention, the period of time sufficient to allow development of a substantially efficacious response to the antimetabolite is between about 4 hours and 24 hours.

As noted above, the combination of an HSP90 inhibitor and an antimetabolite allows for the use of a lower therapeutic dose of the antimetabolite for the treatment of cancer. That a lower dose of antimetabolite is used oftentimes lessens the side effects observed in a subject. The lessened side effects can be measured both in terms of incidence and severity. Severity measures are provided through a grading process delineated by the National Cancer Institute (common toxicity criteria NCI CTC, Version 2). For instance, the incidence of side effects are typically reduced 10%. Oftentimes, the incidence is reduced 20%, 30%, 40% or 50%. Furthermore, the incidence of grade 3 or 4 toxicities for more common side effects associated with antimetabolite administration (e.g., anemia, anorexia, diarrhea, fatigue, nausea and vomiting) is oftentimes reduced 10%, 20%, 30%, 40% or 50%.

Formulations used in the present invention may be in any suitable form, such as a solid, semisolid, or liquid form. See Pharmaceutical Dosage Forms and Drug Delivery Systems, 5^(th) edition, Lippicott Williams & Wilkins (1991), incorporated herein by reference. In general the pharmaceutical preparation will contain one or more of the compounds of the present invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use. The carriers that can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations in solid, semi-solid, or liquefied form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used. Where applicable, the compounds useful in the methods of the invention may be formulated as microcapsules and nanoparticles. General protocols are described, for example, by Microcapsules and Nanoparticles in Medicine and Pharmacy by Max Donbrow, ed., CRC Press (1992) and by U.S. Pat. Nos. 5,510,118, 5,534,270 and 5,662,883 which are all incorporated herein by reference. By increasing the ratio of surface area to volume, these formulations allow for the oral delivery of compounds that would not otherwise be amenable to oral delivery. The compounds useful in the methods of the invention may also be formulated using other methods that have been previously used for low solubility drugs. For example, the compounds may form emulsions with vitamin E or a PEGylated derivative thereof as described by PCT publications WO 98/30205 and WO 00/71163, each of which is incorporated herein by reference. Typically, the compound useful in the methods of the invention is dissolved in an aqueous solution containing ethanol (preferably less than 1% w/v). Vitamin E or a PEGylated-vitamin E is added. The ethanol is then removed to form a pre-emulsion that can be formulated for intravenous or oral routes of administration. Another method involves encapsulating the compounds useful in the methods of the invention in liposomes. Methods for forming liposomes as drug delivery vehicles are well known in the art. Suitable protocols include those described by U.S. Pat. Nos. 5,683,715, 5,415,869, and 5,424,073 which are incorporated herein by reference relating to another relatively low solubility cancer drug paclitaxel and by PCT Publicaton WO 01/10412 which is incorporated herein by reference relating to epothilone B. Of the various lipids that may be used, particularly preferred lipids for making encapsulated liposomes include phosphatidylcholine and polyethyleneglycol-derivatized distearyl phosphatidyl-ethanoloamine.

Yet another method involves formulating the compounds useful in the methods of the invention using polymers such as biopolymers or biocompatible (synthetic or naturally occurring) polymers. Biocompatible polymers can be categorized as biodegradable and non-biodegradable. Biodegradable polymers degrade in vivo as a function of chemical composition, method of manufacture, and implant structure. Illustrative examples of synthetic polymers include polyanhydrides, polyhydroxyacids such as polylactic acid, polyglycolic acids and copolymers thereof, polysters, polyamides, polyorthoesters and some polyphosphazenes. Illustrative examples of naturally occurring polymers include proteins and polysaccharides such as collagen, hyaluronic acid, albumin, and gelatin.

Another method involves conjugating the compounds useful in the methods of the invention to a polymer that enhances aqueous solubility. Examples of suitable polymers include polyethylene glycol, poly-(d-glutamic acid), poly-(1-glutamic acid), poly-(1-glutamic acid), poly-(d-aspartic acid), poly-(1-aspartic acid) and copolymers thereof. Polyglutamic acids having molecular weights between about 5,000 to about 100,000 are preferred, with molecular weights between about 20,000 and 80,000 being more preferred wand with molecular weights between about 30,000 and 60,000 being most preferred. The polymer is conjugated via an ester linkage to one or more hydroxyls of an inventive geldanamycin using a protocol as essentially described by U.S. Pat. No. 5,977,163 which is incorporated herein by reference.

In another method, the compounds useful in the methods of the invention are conjugated to a monoclonal antibody. This method allows the targeting of the inventive compounds to specific targets. General protocols for the design and use of conjugated antibodies are described in Monoclonal Antibody-Based Therapy of Cancer by Michael L. Grossbard, ED. (1998), which is incorporated herein by reference.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. For example, a formulation for intravenous use comprises an amount of the inventive compound ranging from about 1 mg/mL to about 25 mg/mL, preferably from about 5 mg/mL, and more preferably about 10 mg/mL. Intravenous formulations are typically diluted between about 2 fold and about 30 fold with normal saline or 5% dextrose solution prior to use.

Preferably, 17-AAG is formulated as a pharmaceutical solution formulation comprising 17-AAG in an concentration of up to 15 mg/mL dissolved in a vehicle comprising (i) a first component that is ethanol, in an amount of between about 40 and about 60 volume %; (ii) a second component that is a polyethoxylated castor oil, in an amount of between about 15 to about 50 volume %; and (iii) a third component that is selected from the group consisting of propylene glycol, PEG 300, PEG 400, glycerol, and combinations thereof, in an amount of between about 0 and about 35 volume %. The aforesaid percentages are volume/volume percentages based on the combined volumes of the first, second, and third components. The lower limit of about 0 volume % for the third component means that it is an optional component; that is, it may be absent. The pharmaceutical solution formulation is then diluted into water to prepare a diluted formulation containing up to 3 mg/mL 17-AAG, for intravenous formulation.

Preferably, the second component is Cremophor EL and the third component is propylene glycol. In an especially preferred formulation, the percentages of the first, second, and third components are 50%, 20-30%, and 20-30%, respectively.

Other formulations designed for 17-AAG are described in Tabibi et al., U.S. Pat. No. 6,682,758 B1 (2004) and Ulm et al., WO 03/086381 A1 (2003); the disclosures of which are incorporated herein by reference.

The method of the present invention is used for the treatment of cancer. In one embodiment, the methods of the present invention are used to treat cancers of the head and neck, which include, but are not limited to, tumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas. In another embodiment, the compounds of the present invention are used to treat cancers of the liver and biliary tree, particularly hepatocellular carcinoma. In another embodiment, the compounds of the present invention are used to treat intestinal cancers, particularly colorectal cancer. In another embodiment, the compounds of the present invention are used to treat ovarian cancer. In another embodiment, the compounds of the present invention are used to treat small cell and non-small cell lung cancer. In another embodiment, the compounds of the present invention are used to treat breast cancer. In another embodiment, the compounds of the present invention are used to treat sarcomas, including fibrosarcoma, malignant fibrous histiocytoma, embryonal rhabdomysocarcoman, leiomysosarcoma, neuro-fibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, and alveolar soft part sarcoma. In another embodiment, the compounds of the present invention are used to treat neoplasms of the central nervous systems, particularly brain cancer. In another embodiment, the compounds of the present invention are used to treat lymphomas which include Hodgkin's lymphoma, lymphoplasmacytoid lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, mantle cell lymphoma, B-lineage large cell lymphoma, Burkitt's lymphoma, and T-cell anaplastic large cell lymphoma.

EXAMPLES

The following Examples are provided to illustrate certain aspects of the present invention and to aid those of skill in the art in practicing the invention.

Materials and Methods

Cell Line and Reagents

Human colon adenocarcinoma cell line, DLD-1, and human breast adenocarcinoma cell line, SKBr-3, were obtained from American Type Culture Collection (manassas, Va.). DLD-1 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, and SKBr-3 cells were cultured in McCoy's 5a medium supplemented with 10% fetal bovine serum. 17-DMAG and 17-AAG were obtained using published procedures. Other cytotoxic agents were purchased commercially from suppliers such as Sigma Chemical Co. (St. Louis, Mo.) and Sequoia Research Products (Oxford, UK).

Cell Viability Assay and Combination Effect Analysis

Cells were seeded in duplicate in 96-well microtiter plates at a density of 5,000 cells per well and allowed to attach overnight. Cells were treated with 17-AAG or 17-DMAG and the corresponding cytotoxic drug at varying concentrations, ranging from 0.5 picomolar (“pM”) to 50 micromolar (“μM”), for 3 days. Cell viability was determined using the MTS assay (Promega). For the drug combination assay, cells were seeded in duplicate in 96-well plates (5,000 cells/well). After an overnight incubation, cells were treated with drug alone or a combination and the IC₅₀ value (the concentration of drug required to inhibit cell growth by 50%) was determined. Based on the IC₅₀ values of each individual drug, combined drug treatment was designed at constant ratios of two drugs, i.e., equivalent to the ratio of their IC₅₀. Two treatment schedules were used: In one schedule, the cells were exposed to 24 hours of 17-AAG or 17-DMAG. The drug was then added to the cells and incubated for 48 hours. In another schedule, cells were exposed to the drug alone for 24 hours followed by addition of 17-AAG or 17-DMAG for 48 hours. Cell viability was determined by the MTS assay.

Synergism, additivity or antagonism was determined by median effect analysis using the combination index (CI) calculated using Calcusyn (Biosoft, Cambridge, UK). The combination index is defined as follows: CI=[D] ₁ /[D _(x)]₁ +[D] ₂ /[D _(x)]₂ The quantities [D]₁ and [D]₂ represent the concentrations of the first and second drug, respectively, that in combination provide a response of x % in the assay. The quantities [D_(x)]₁ and [D_(x)]₂ represent the concentrations of the first and second drug, respectively, that when used alone provide a response of x % in the assay. Values of CI<1, CI=1, and CI>1 indicated drug-drug synergism, additivity, and antagonism respectively (Chou and Talalay 1984). The “enhancing” effect of two drugs can also be determined. Results 17-AAG Combination in DLD-1 Cells

The following table provides CI values for combinations of 17-AAG and the antimetabolites 5-fluorouracil, gemicitabine and methotrexate in a DLD-1 cell assay. “Pre-administration” refers to the administration of 17-AAG to the cells before the administration of antimetabolite; “post-administration” refers to the administration of 17-AAG to the cells after the administration of antimetabolite. TABLE 5 CI values for combinations in DLD-1 cells (human colorectal cancer cells). 17-AAG Antimetabolite 17-AAG Pre-Administration Post-Administration 5-Fluorouracil 0.96 ± 0.16 0.33 ± 0.08 Gemcitabine 0.45 ± 0.19 0.77 ± 0.23 Methotrexate 0.83 ± 0.14 0.71 ± 0.35 17-AAG Combination in SKSBr-3 Cells

The following table provides CI values for combinations of 17-AAG and the antimetabolites 5-fluorouracil and gemicitabine in an SKBr-3 cell assay. TABLE 6 CI values for combinations in SKBr cells (human breast cancer cells). 17-AAG Antimetabolite 17-AAG Pre-Administration Post-Administration 5-Fluorouracil 0.65 ± 0.48 0.1.01 ± 0.13   Gemcitabine 1.79 ± 0.54 0.86 ± 0.19 Additional Observations

Additional analysis indicated that both 17-AAG and 17-DMAG reduced the expression of ErbB2 protein in SKBr3 and glioma cells. This observation, taken in combination with the results reported above, indicates that combinations of 17-AAG or 17-DMAG with any of the antimetabolites above that are known to be useful to treat diseases characterized by elevated ErbB2 protein expression (i.e., levels of expressions of ErbB2 protein greater than those found in healthy cells). Similarly, combinations of 17-AAG and 5-FU reduced the expression of Raf-1 and Src kinase proteins, also demonstrating that this combination is especially effective in treating diseases characterized by elevated expression of these two proteins. 

1. A method for treating colon cancer in a patient, wherein the method comprises administering an HSP90 inhibitor and an antimetabolite to the patient.
 2. The method of claim 1, wherein the HSP90 inhibitor is administered to the patient before the antimetabolite.
 3. The method of claim 1, wherein the HSP90 inhibitor is administered to the patient after the antimetabolite.
 4. The method of claim 1, wherein the antimetabolite is a purine analog.
 5. The method of claim 1, wherein the antimetabolite is selected from a group of antimetabolites consisting of methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine monophosphate, cytarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine.
 6. The method of claim 2, wherein the antimetabolite is 5-fluorouracil.
 7. The method of claim 3, wherein the antimetabolite is gemcitabine.
 8. The method of claim 6, wherein the HSP90 inhibitor is geldanamycin or a geldanamycin derivative.
 9. The method of claim 7, wherein the HSP90 inhibitor is geldanamycin or a geldanamycin derivative.
 10. The method of claim 8, wherein the HSP90 inhibitor is a geldanamycin derivative, and wherein the derivative is 17-AAG.
 11. The method of claim 9, wherein the HSP90 inhibitor is a geldanamycin derivative, and wherein the derivative is 17-AAG.
 12. The method of claim 10, wherein the therapeutic dose of 5-fluorouracil in a multiple-weekly regimen is less than 12 mg/kg.
 13. The method of claim 11, wherein the therapeutic dose of gemcitabine in a once-weekly regimen is less than 1000 mg/m².
 14. The method of claim 12, wherein the therapeutic dose of 5-fluorouracil in a multiple-weekly regimen is less than 11 mg/kg.
 15. The method of claim 13, wherein the therapeutic dose of gemcitabine in a once-weekly regimen is less than 950 mg/m².
 16. The method of claim 14, wherein the therapeutic dose of 5-fluorouracil in a multiple-weekly regimen is less than 10 mg/kg.
 17. The method of claim 15, wherein the therapeutic dose of gemcitabine in a once-weekly regimen is less than 900 mg/m².
 18. The method of claim 1, wherein the HSP90 inhibitor is 17-AAG, and wherein the administration of 17-AAG and the antimetabolite is performed once per week.
 19. The method of claim 1, wherein the HSP90 inhibitor is 17-AAG, and wherein the administration of 17-AAG and the antimetabolite is performed twice per week.
 20. The method of claim 18, wherein the therapeutic dose of 17-AAG is between 50 mg/m² and 450 mg/m².
 21. The method of claim 19, wherein the therapeutic dose of 17-AAG is between 50 mg/m² and 250 mg/m².
 22. The method of claim 20, wherein the therapeutic dose of 17-AAG is between 150 mg/m² and 350 mg/m².
 23. The method of claim 21, wherein the therapeutic dose of 17-AAG is between 150 mg/m² and 250 mg/m².
 24. The method of claim 22, wherein the therapeutic dose of 17-AAG is about 308 mg/m².
 25. The method of claim 23, wherein the therapeutic dose of 17-AAG is about 220 mg/m². 